Capillary zone electrophoresis ("CZE") in small bore capillaries was first demonstrated by Jorgenson and Lukacs, and has proven useful as an efficient method for the separation of certain small solutes. J. Chromatog., 218 (1981), 209; Anal. Chem., 53 (1981), 1298. In CZE, an electric field is applied between the two ends of a capillary tube into which an electrolyte containing the solutes is introduced. The electric field causes the electrolyte to flow through the tube. Some solutes will have higher electrokinetic mobilities than other solutes so that the sample components are resolved into zones in the capillary tube during the flow of the electrolytes through the capillary.
Attractive factors for CZE include the small sample sizes, high resolution, automation, and the potential for quantification and recovery of biologically active samples. For example, U.S. Pat. No. 4,675,300, inventors Zare et al., describes theories and equipment for electrokinetic separation processes employing a laser-excited fluorescence detector. The system described by Zare et al. includes a fused silica capillary with a 75.mu. inner diameter. CZE has been increasingly used in the analysis of a variety of substances, such as amino acids, proteins, nucleotides, nucleosides and drugs.
Jorgenson and Lukacs reported problems associated with the separation of proteins. It was found that most of proteins exhibit significant adsorption to the surface of both fused silica and borosilicate glass capillaries. They concluded that adsorption affects electropherograms in two undesirable ways. First, it leads to broad asymmetric "tailed" zones. Second, adsorbed proteins modified the capillary surface, usually decreasing electroosmotic flow significantly which leads to unpredictable migration for all sample zones upon repeated injection. Jorgenson and Lukacs, Science, 222 (1983), 266.
Lauer and McManigill, Anal. Chem., 58 (1986), 166, have reported that the Coulombic repulsion between proteins and the capillary wall of silica capillaries can overcome adsorption tendencies of the proteins with the capillary wall. They demonstrated separations of model proteins (ranging in molecular weight from 13,000 to 77,000) by varying the solution pH relative to the isoelectric point (pI) of the proteins to change their net charge. Several other approaches have employed to eliminate the wall absorption of proteins: applying very low pH values so that the silanol groups are largely protonated and result in a small electrical charge on the wall; and chemical modification of the wall with a neutral, hydrophilic moiety in order shield the silanol groups. See Swedberg, "Characterization of Protein Behavior in High-Performance Capillary Electrophoresis Using a Novel Capillary System", Anal. Biochem., 185 (1990) 51; Bruin et al., "Performance of Carbohydrate-Modified Fused-Silica Capillaries for the Separation of Proteins by Zone Electrophoresis", J. Chromatogr., 480 (1989), 339; Bruin, et al., "Capillary Zone Electrophoretic Separations of proteins in Polyethylene Glycol-Modified Capillaries", J. Chromatogr., 471 (1989), 429; McCormick, "Capillary Zone Electrophoretic Separation of Peptides and Proteins Using Low pH Buffers in Modified Silica Capillaries", Anal. Chem., 60 (1988), 2322; and Hjerten, "High-Performance Electrophoresis Elimination of Electroendosmosis and Solute Adsorption", J. Chromatogr., 347 (1985), 191.
Increasing the selectivity control of capillary electrophoresis has been achieved through the use of anionic micelles from sodium dodecyl sulfate (SDS). This approach has been used to separate bases, nucleosides and nucleotides in a buffer solution with a pH of 7. Since the bases and nucleosides are uncharged at the pH of operation, separation is a result of differential partition within the interior of the micelle; the more hydrophobic the species, the larger the partition coefficient and the larger the retention. Oligo-nucleotides are negatively charged and can be separated without SDS micelles; however, the time window is narrow and separation of complex mixtures is limited. The combination of low concentrations of divalent metals and SDS micelles leads to a significant enhancement of the time window and good separation of oligonucleotides. The metal ion is electrostatically attracted to the surface of the micelle and differential metal complexation of the oligonucleotides with the surface of micelles leads to separation of complex mixtures. See Cohen, Anal. Chem., 59 (1987), 1021.
Traditional analysis of species in a complex matrix requires pretreatment steps such as extraction or precipitation to partially clean the sample to rid it of interferants. See Roach et al., J. Chromatogr., 426 (1988), 129. However, pretreatment increases analysis time and increases the probability of contamination and of the inadvertent elimination of solutes from analysis.